Process for the Production of Immunogenic Compositions

ABSTRACT

The present invention relates to a process for producing immunogenic polypeptides, comprising reducing disulfide bonds and blocking the resulting free thiol group with a blocking agent. The immunogenic peptides comprise a fragment of MAGE A3.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of pending U.S. Ser. No. 09/601,565filed 3 Aug. 2000 (the contents of which are incorporated by referenceherein), which is a 371 of PCT/EP99/006600 filed 2 Feb. 1999 (thecontents of which are incorporated by reference herein). Thisapplication also claims priority to Great Britain applicationsGB9802543.0 filed on 5 Feb. 1998 and GB9802650.3 filed on 6 Feb. 1998.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable

BACKGROUND OF THE INVENTION

The present invention relates to protein derivatives, comprising atumor-associated antigen, that find utility in cancer vaccine therapy.In particular the derivatives of the invention include fusion proteinscomprising an antigen encoded by the family of MAGE genes (e.g. MAGE-3,MAGE-1), linked to an immunological fusion partner which provides Thelper epitopes, such as, for example the lipidated form of protein Dfrom Haemophilus influenzae B; chemically modified MAGE proteins whereinthe antigen's disulphide bridges are reduced and the resulting thiolsblocked and genetically modified MAGE proteins provided with an affinitytag and/or genetically modified to prevent disulphide bridge formation.Methods are also described for purifying MAGE proteins and forformulating vaccines for treating a range of cancers, including, but notlimited to Melanoma, breast, bladder, lung, NSCLC, head and squamouscell carcinoma, colon carcinoma and oesophagus carcinoma.

Antigens encoded by the family of MAGE genes are predominately expressedon melanoma cells (including malignant melanoma) and some other cancersincluding NSCLC (non small cell lung cancer), head and neck squamouscell carcinoma, bladder transitional cell carcinoma and oesophaguscarcinoma, but are not detectable on normal tissues except in the testisand the placenta (Gaugler, 1994; Weynants, 1994; Patard, 1995). MAGE-3is expressed in 69% of melanomas (Gaugler, 1994), and can also bedetected in 44% of NSCLC (Yoshimatsu 1988), 48% of head and necksquamous cell carcinoma, 34% of bladder transitional cell carcinoma 57%of oesophagus carcinoma 32% of colon cancers and 24% of breast cancers(Van Pel, 1995); Inoue, 1995 Fujie 1997; Nishimura 1997). Cancersexpressing MAGE proteins are known as Mage associated tumours.

The immunogenicity of human melanoma cells has been elegantlydemonstrated in experiments using mixed cultures of melanoma cells andautologous lymphocytes. These culture often generate specific cytotoxicT lymphocytes (CTLs) able to lyse exclusively the autologous melanomacells but neither autologous fibroblasts, nor autologous EBV-transformedB lymphocytes (Knuth, 1984; Anichini, 1987). Several of the antigensrecognised on autologous melanoma cells by these CTL clones are nowidentified, including those of the MAGE family.

The first antigen which could be defined through its recognition byspecific CTLs on autologous melanoma cells is termed MZ2-E (Van denEynde, 1989) and is encoded by the gene MAGE-1 (Van der Bruggen, 1991).CTLs directed against MZ2-E recognise and lyse MZ2-E positive melanomacells from autologous as well as from other patients provided that thesecells have the HLA.A1 allele.

The MAGE-1 gene belongs to a family of 12 closely related genes, MAGE 1,MAGE 2, MAGE 3, MAGE 4, MAGE 5, MAGE 6, MAGE 7, MAGE 8, MAGE 9, MAGE 10,MAGE 11, MAGE 12, located on chromosome X and sharing with each other 64to 85% homology in their coding sequence (De Plaen, 1994). These aresometimes known as MAGE A1, MAGE A2, MAGE A3, MAGE A4, MAGE A5, MAGE A6,MAGE A7, MAGE A8, MAGE A9, MAGE A 10, MAGE All, MAGE A 12 (The MAGE Afamily). Two other groups of proteins are also part of the MAGE familyalthough more distantly related. These are the MAGE B and MAGE C group.The MAGE B family includes MAGE B1 (also known as MAGE Xp1, and DAM 10),MAGE B2 (also known as MAGE Xp2 and DAM 6) MAGE B3 and MAGE B4—the MageC family currently includes MAGE C1 and MAGE C2. In general terms, aMAGE protein can be defined as containing a core sequence signaturelocated towards the C-terminal end of the protein (for example withrespect to MAGE A1 a 309 amino acid protein, the core signaturecorresponds to amino acid 195-279).

The consensus pattern of the core signature is thus described as followswherein x represents any amino acid, lower case residues are conserved(conservative variants allowed) and upper case residues are perfectlyconserved.

Core Sequence Signature

(SEQ ID NO: 16) LixvL(2x)I(3x)g(2x)apEExiWexl(2x)m(3-4x)Gxe(3-4x)gxp(2x)llt(3x)VqexYLxYxqVPxsxP(2x)yeFLWGprA(2x) Et(3x)kv

Conservative substitutions are well known and are generally set up asthe default scoring matrices in sequence alignment computer programs.These programs include PAM250 (Dayhoft M. O. et al., (1978), “A model ofevolutionary changes in proteins”, In “Atlas of Protein sequence andstructure” 5(3) M. O. Dayhoft (ed.), 345-352), National BiomedicalResearch Foundation, Washington, and Blosum 62 (Steven Henikoft andJorja G. Henikoft (1992), “Amino acid substitution matricies fromprotein blocks”), Proc. Natl. Acad. Sci. USA 89 (Biochemistry):10915-10919.

In general terms, substitution within the following groups areconservative substitutions, but substitutions between groups areconsidered non-conserved. The groups are:

i) Aspartate/asparagine/glutamate/glutamine

ii) Serine/threonine

iii) Lysine/arginine

iv) Phenylalanine/tyrosine/tryptophane

v) Leucine/isoleucine/valine/methionine

vi) Glycine/alanine

In general and in the context of this invention, a MAGE protein will beapproximately 50% identical in this core region with amino acids 195 to279 of MAGE A1.

Several CTL epitopes have been identified on the MAGE-3 protein. Onesuch epitope, MAGE-3.A1, is a nonapeptide sequence located between aminoacids 168 and 176 of the MAGE-3 protein which constitutes an epitopespecific for CTLs when presented in association with the MHC class Imolecule HLA.A1. Recently two additional CTL epitopes have beenidentified on the peptide sequence of the MAGE-3 protein by theirability to mount a CTL response in a mixed culture of melanoma cells andautologous lymphocytes. These two epitopes have specific binding motifsfor the HLA.A2 (Van der Bruggen, 1994) and HLA.B44 (Herman, 1996)alleles respectively.

BRIEF SUMMARY OF THE INVENTION

The present invention provides MAGE protein derivatives. Suchderivatives are suitable for use in therapeutic vaccine formulationswhich are suitable for the treatment of a range of tumour types.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: LPD-MAGE-3-His

FIG. 2: Construction of the Expression Vector pRIT 14586

FIG. 3: Construction of the plasmid pRIT 14477 expressing the fusionprotein Prot. D 1/3 MAGE-3-His tail

FIG. 4: Western blot analysis of LPD-MAGE-3-His protein Anti-MAGE-3monoclonal antibodies Mab 32 and Mab 54

FIG. 5. Immunogenicity of MAGE-3 in mice (C57BL6)

FIG. 6: Immunogenicity of MAGE-3 in mice (C57BL6)

FIG. 7: Immunogenicity of MAGE-3 in mice (BalbC)

FIG. 8: Immunogenicity of MAGE-3 in mice (BalbC)

FIG. 9: Anti-Mage antibodies in the serum of mice immunized with LipoDMage3 His in SBAS2 or not

FIG. 10: Subclass-specific antibody responses in Balc/b mice

FIG. 11: Subclass-specific antibody responses in C57BL/C mice

FIG. 12: NS-Asp-MAGE-3-Gly-Gly-7Xhis

FIG. 13: Construction of plasmid pRIT14426

FIG. 14: Plasmid map of pRIT14426

FIG. 15: CLYTA-Asp-Met-Gly-MAGE-1-Gly-Gly-His(7)

FIG. 16: Construction of plasmid pRIT14613

FIG. 17: Construction of plasmid pRIT

FIG. 18: CLYTA-Asp-Ser-Met-Leu-Asp-MAGE-3-Gly-Gly-His(7)

FIG. 19: Construction of plasmid pRIT 14646

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment of the present invention, the derivative is a fusionproteins comprising an antigen from the MAGE protein family linked to aheterologous partner. The proteins may be chemically conjugated, but arepreferably expressed as recombinant fusion proteins allowing increasedlevels to be produced in an expression system as compared to non-fusedprotein. Thus the fusion partner may assist in providing T helperepitopes (immunological fusion partner), preferably T helper epitopesrecognised by humans, or assist in expressing the protein (expressionenhancer) at higher yields than the native recombinant protein.Preferably the fusion partner will be both an immunological fusionpartner and expression enhancing partner.

In a preferred form of the invention, the immunological fusion partneris derived from protein D, a surface protein of the gram-negativebacterium, Haemophilus influenza B (WO91/18926). Preferably the proteinD derivative comprises approximately the first 1/3 of the protein, inparticular approximately the first N-terminal 100-110 amino acids.Preferably the protein D derivative is lipidated. Preferably the first109 residues of the Lipoprotein D fusion partner is included on theN-terminus to provide the vaccine candidate antigen with additionalexogenous T-cell epitopes and increase expression level in E-coli (thusacting also as an expression enhancer). The lipid tail ensures optimalpresentation of the antigen to antigen presenting cells.

Other fusion partners include the non-structural protein from influenzaevirus, NS1 (hemagglutinin). Typically the N terminal 81 amino acids areutilised, although different fragments may be used provided they includeT-helper epitopes.

In another embodiment the immunological fusion partner is the proteinknown as LYTA. Preferably the C terminal portion of the molecule isused. Lyta is derived from Streptococcus pneumoniae which synthesize anN-acetyl-L-alanine amidase, amidase LYTA, (coded by the lytA gene {Gene,43 (1986) page 265-272} an autolysin that specifically degrades certainbonds in the peptidoglycan backbone. The C-terminal domain of the LYTAprotein is responsible for the affinity to the choline or to somecholine analogues such as DEAE. This property has been exploited for thedevelopment of E. coli C-LYTA expressing plasmids useful for expressionof fusion proteins. Purification of hybrid proteins containing theC-LYTA fragment at its amino terminus has been described {Biotechnology:10, (1992) page 795-798}. As used herein a preferred embodiment utilisesthe repeat portion of the Lyta molecule found in the C terminal endstarting at residue 178. A particularly preferred form incorporatesresidues 188-305.

The immunological fusion partners noted above are also advantageous inaiding expression. In particular, such fusions are expressed at higheryields than native recombinant MAGE proteins.

Such constructs in a clinical setting have been shown by the presentinventors to be able to treat melanoma. In one case, a patient withstage 1V melanoma was cleared of metasties after two doses ofunadjuvanted lipo D 1/3 MAGE 3 His protein.

Accordingly, the present invention in the embodiment provides fusionproteins comprising a tumour-associated antigen from the MAGE familylinked to an immunological fusion partner. Preferably the immunologicalfusion partner is protein D or fragment thereof, most preferablylipoprotein D. The MAGE proteins are preferably MAGE A1 or MAGE A3. TheLipoprotein D part preferably comprises the first 1/3 of Lipoprotein D.

The proteins of the present invention preferably are expressed in E.coli. In a preferred embodiment the proteins are expressed with anaffinity tag, such as for example, a histidine tail comprising between 5to 9 and preferably six histidine residues. These are advantageous inaiding purification.

The present invention also provides a nucleic acid encoding the proteinsof the present invention. Such sequences can be inserted into a suitableexpression vector and used for DNA/RNA vaccination or expressed in asuitable host. Microbial vectors expressing the nucleic acid may be usedas vaccines. Such vectors include for example, poxvirus, adenovirus,alphavirus, listeria and monarphage.

A DNA sequence encoding the proteins of the present invention can besynthesized using standard DNA synthesis techniques, such as byenzymatic ligation as described by D. M. Roberts et al. in Biochemistry1985, 24, 5090-5098, by chemical synthesis, by in vitro enzymaticpolymerization, or by PCR technology utilising for example a heat stablepolymerase, or by a combination of these techniques.

Enzymatic polymerisation of DNA may be carried out in vitro using a DNApolymerase such as DNA polymerase I (Klenow fragment) in an appropriatebuffer containing the nucleoside triphosphates dATP, dCTP, dGTP and dTTPas required at a temperature of 10°-37° C., generally in a volume of 50μl or less. Enzymatic ligation of DNA fragments may be carried out usinga DNA ligase such as T4 DNA ligase in an appropriate buffer, such as0.05M Tris (pH 7.4), 0.01M MgCl₂, 0.01M dithiothreitol, 1 mM spermidine,1 mM ATP and 0.1 mg/ml bovine serum albumin, at a temperature of 4° C.to ambient, generally in a volume of 50 ml or less. The chemicalsynthesis of the DNA polymer or fragments may be carried out byconventional phosphotriester, phosphite or phosphoramidite chemistry,using solid phase techniques such as those described in ‘Chemical andEnzymatic Synthesis of Gene Fragments—A Laboratory Manual’ (ed. H. G.Gassen and A. Lang), Verlag Chemie, Weinheim (1982), or in otherscientific publications, for example M. J. Gait, H. W. D. Matthes, M.Singh, B. S. Sproat, and R. C. Titmas, Nucleic Acids Research, 1982, 10,6243; B. S. Sproat, and W. Bannwarth, Tetrahedron Letters, 1983, 24,5771; M. D. Matteucci and M. H. Caruthers, Tetrahedron Letters, 1980,21, 719; M. D. Matteucci and M. H. Caruthers, Journal of the AmericanChemical Society, 1981, 103, 3185; S. P. Adams et al., Journal of theAmerican Chemical Society, 1983, 105, 661; N. D. Sinha, J. Biernat, J.McMannus, and H. Koester, Nucleic Acids Research, 1984, 12, 4539; and H.W. D. Matthes et al., EMBO Journal, 1984, 3, 801.

The process of the invention may be performed by conventionalrecombinant techniques such as described in Maniatis et al., MolecularCloning—A Laboratory Manual; Cold Spring Harbor, 1982-1989.

In particular, the process may comprise the steps of:

i) preparing a replicable or integrating expression vector capable, in ahost cell, of expressing a DNA polymer comprising a nucleotide sequencethat encodes the protein or an immunogenic derivative thereof;

ii) transforming a host cell with said vector;

iii) culturing said transformed host cell under conditions permittingexpression of said DNA polymer to produce said protein; and

iv) recovering said protein.

The term ‘transforming’ is used herein to mean the introduction offoreign DNA into a host cell. This can be achieved for example bytransformation, transfection or infection with an appropriate plasmid orviral vector using e.g. conventional techniques as described in GeneticEngineering; Eds. S. M. Kingsman and A. J. Kingsman; BlackwellScientific Publications; Oxford, England, 1988. The term ‘transformed’or ‘transformant’ will hereafter apply to the resulting host cellcontaining and expressing the foreign gene of interest.

The expression vectors are novel and also form part of the invention.

The replicable expression vectors may be prepared in accordance with theinvention, by cleaving a vector compatible with the host cell to providea linear DNA segment having an intact replicon, and combining saidlinear segment with one or more DNA molecules which, together with saidlinear segment encode the desired product, such as the DNA polymerencoding the protein of the invention, or derivative thereof, underligating conditions.

Thus, the DNA polymer may be preformed or formed during the constructionof the vector, as desired.

The choice of vector will be determined in part by the host cell, whichmay be prokaryotic or eukaryotic but are preferably E. Coli or CHOcells. Suitable vectors include plasmids, bacteriophages, cosmids andrecombinant viruses.

The preparation of the replicable expression vector may be carried outconventionally with appropriate enzymes for restriction, polymerisationand ligation of the DNA, by procedures described in, for example,Maniatis et al. cited above.

The recombinant host cell is prepared, in accordance with the invention,by transforming a host cell with a replicable expression vector of theinvention under transforming conditions. Suitable transformingconditions are conventional and are described in, for example, Maniatiset al. cited above, or “DNA Cloning” Vol. II, D. M. Glover ed., IRLPress Ltd, 1985.

The choice of transforming conditions is determined by the host cell.Thus, a bacterial host such as E. coli may be treated with a solution ofCaCl₂ (Cohen et al., Proc. Nat. Acad. Sci., 1973, 69, 2110) or with asolution comprising a mixture of RbCl, MnCl₂, potassium acetate andglycerol, and then with 3-[N-morpholino]-propane-sulphonic acid, RbCland glycerol. Mammalian cells in culture may be transformed by calciumco-precipitation of the vector DNA onto the cells. The invention alsoextends to a host cell transformed with a replicable expression vectorof the invention.

Culturing the transformed host cell under conditions permittingexpression of the DNA polymer is carried out conventionally, asdescribed in, for example, Maniatis et al. and “DNA Cloning” citedabove. Thus, preferably the cell is supplied with nutrient and culturedat a temperature below 50° C.

The product is recovered by conventional methods according to the hostcell and according to the localisation of the expression product(intracellular or secreted into the culture medium or into the cellperiplasm). Thus, where the host cell is bacterial, such as E. coli itmay, for example, be lysed physically, chemically or enzymatically andthe protein product isolated from the resulting lysate. Where the hostcell is mammalian, the product may generally be isolated from thenutrient medium or from cell free extracts. Conventional proteinisolation techniques include selective precipitation, adsorptionchromatography, and affinity chromatography including a monoclonalantibody affinity column.

The proteins of the present invention are provided either soluble in aliquid form or in a lyophilised form.

It is generally expected that each human dose will comprise 1 to 1000 μgof protein, and preferably 30-300 μg.

The present invention also provides pharmaceutical compositioncomprising a protein of the present invention in a pharmaceuticallyacceptable excipient.

A preferred vaccine composition comprises at least Lipoprotein D-MAGE-3.Such vaccine may optionally contain one or more other tumor-associatedantigen. For example other members belonging to the MAGE and GAGEfamilies. Suitable other tumour associated antigen include MAGE-1,GAGE-1 or Tyrosinase proteins.

Vaccine preparation is generally described in Vaccine Design (“Thesubunit and adjuvant approach” (eds. Powell M. F. & Newman M. J). (1995)Plenum Press New York). Encapsulation within liposomes is described byFullerton, U.S. Pat. No. 4,235,877.

The proteins of the present invention are preferably adjuvanted in thevaccine formulation of the invention. Suitable adjuvants include analuminium salt such as aluminium hydroxide gel (alum) or aluminiumphosphate, but may also be a salt of calcium, iron or zinc, or may be aninsoluble suspension of acylated tyrosine, or acylated sugars,cationically or anionically derivatised polysaccharides, orpolyphosphazenes. Other known adjuvants include CpG containingoligonucleotides. The oligonucleotides are characterised in that the CpGdinucleotide is unmethylated. Such oligonucleotides are well known andare described in, for example WO96/02555.

In the formulation of the inventions it is preferred that the adjuvantcomposition induces an immune response preferentially of the TH1 type.Suitable adjuvant systems include, for example a combination ofmonophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipidA (3D-MPL) together with an aluminium salt. CpG oligonucleotides alsopreferentially induce a TH1 response.

An enhanced system involves the combination of a monophosphoryl lipid Aand a saponin derivative particularly the combination of QS21 and 3D-MPLas disclosed in WO 94/00153, or a less reactogenic composition where theQS21 is quenched with cholesterol as disclosed in WO 96/33739.

A particularly potent adjuvant formulation involving QS21 3D-MPL &tocopherol in an oil in water emulsion is described in WO 95/17210 andis a preferred formulation.

Accordingly in one embodiment of the present invention there is provideda vaccine comprising a protein of the present invention, more preferablya Lipoprotein D (or derivative thereof)—MAGE-3 adjuvanted with amonophosphoryl lipid A or derivative thereof.

Preferably the vaccine additionally comprises a saponin, more preferablyQS21.

Preferably the formulation additional comprises an oil in water emulsionand tocopherol. The present invention also provides a method forproducing a vaccine formulation comprising mixing a protein of thepresent invention together with a pharmaceutically acceptable excipient,such as 3D-MPL.

In one aspect of the invention there is provided a process for purifyinga recombinantly produced MAGE-protein. The process comprisessolubilising the protein, for example in a strong chaotropic agent (suchas for example, urea, guanidium hydrochloride), or in a Zwitterionicnicdetergent, e.g. (Empigen BB—n-dodecyl-N,N-dimethylglycine), reducing theprotein's intra and inter molecular disulphide bonds, blocking theresulting thiols to prevent oxidative recoupling, and subjecting theprotein to one or more chromatographic steps.

Preferably, the blocking agent is an alkylating agent. Such blockingagents include but are not limited to alpha haloacids or alphahaloamides. For example iodoacetic acid and iodoacetamide which resultsin carboxymethylation or carboxyamidation (carbamidomethylation) of theprotein. Other blocking agents may be used and are described in theliterature (See for example, The Proteins Vol II Eds H neurath, R L Hilland C-L Boeder, Academic press 1976, or Chemical Reagents for Proteinmodification Vol I eds. R L Lundblad and C M Noyes, CRC Press 1985).Typical examples of such other blocking agents include N-ethylmaleimide,chloroacetyl phosphate, O-methylisourea and acrylonitrile. The use ofthe blocking agent is advantageous as it prevents aggregation of theproduct, and ensure stability for downstream purification.

In an embodiment of the invention the blocking agents are selected toinduce a stable covalent and irreversible derivative (eg alpha haloacids or alpha haloamides). However other blocking agents maybe selectedsuch that after purification the blocking agent may be removed torelease the non derivatised protein.

MAGE proteins having derivatised free thiol residues are new and form anaspect of the invention. In particular carboxyamidated orcarboxymethylated derivatives are a preferred embodiment of theinvention.

In a preferred embodiment of the invention the proteins of the presentinvention is provided with an affinity tag, such as CLYTA or apolyhistidine tail. In such cases the protein after the blocking step ispreferably subjected to affinity chromatography. For those proteins witha polyhistidine tail, immobilised metal ion affinity chromatography(IMAC) may be performed. The metal ion, may be any suitable ion forexample zinc, nickel, iron, magnesium or copper, but is preferably zincor nickel. Preferably the IMAC buffer contain a zwitterionic detergentsuch as Empigen BB (hereinafter Empigen) as this results in lower levelsof endotoxin in the final product.

If the protein is produced with a Clyta part, the protein may bepurified by exploiting its affinity to choline or choline analogues suchas DEAE. In an embodiment of the invention the proteins are providedwith a polyhistidine tail and a Clyta part. These may purified in asimple two step affinity chromatographic purification schedule.

The invention will be further described by reference to the followingexamples:

Example I Preparation of the Recombinant E. coli Strain Expressing theFusion Protein Lipoprotein D-MAGE-3-His (LPD 1/3-MAGE-3-His or LpDMAGE-3His)

1. The E. coli Expression System:

For the production of Lipoprotein D the DNA encoding protein D has beencloned into the expression vector pMG 81. This plasmid utilizes signalsfrom lambda phage DNA to drive the transcription and translation ofinserted foreign genes. The vector contains the lambda PL promoter PL,operator OL and two utilization sites (NutL and NutR) to relievetranscriptional polarity effects when N protein is provided (Gross etal., 1985. Mol. & Cell. Biol. 5:1015). Vectors containing the PLpromoter, are introduced into an E. coli lysogenic host to stabilize theplasmid DNA. Lysogenic host strains contain replication-defective lambdaphage DNA integrated into the genome (Shatzman et al., 1983; InExperimental Manipulation of Gene Expression. Inouya (ed) pp 1-14.Academic Press NY). The lambda phage DNA directs the synthesis of the cIrepressor protein which binds to the OL repressor of the vector andprevents binding of RNA polymerase to the PL promoter and therebytranscription of the inserted gene. The cI gene of the expression strainAR58 contains a temperature sensitive mutation so that PL directedtranscription can be regulated by temperature shift, i.e. an increase inculture temperature inactivates the repressor and synthesis of theforeign protein is initiated. This expression system allows controlledsynthesis of foreign proteins especially of those that may be toxic tothe cell (Shimataka & Rosenberg, 1981. Nature 292:128).

2. The E. coli Strain AR58:

The AR58 lysogenic E. coli strain used for the production of theLPD-MAGE-3-His protein is a derivative of the standard NIH E. coli K12strain N99 (F-su-galK2, lacZ-thr-). It contains a defective lysogeniclambda phage (galE::TN10, 1 Kil-cI857 DH1). The Kil-phenotype preventsthe shut off of host macromolecular synthesis. The cI857 mutationconfers a temperature sensitive lesion to the cI repressor. The DH1deletion removes the lambda phage right operon and the hosts bio, uvr3,and chlA loci. The AR58 strain was generated by transduction of N99 witha P lambda phage stock previously grown on an SA500 derivative(galE::TN10, 1 Kil-cI857 DH1). The introduction of the defective lysogeninto N99 was selected with tetracycline by virtue of the presence of aTN10 transposon coding for tetracyclin resistance in the adjacent galEgene. N99 and SA500 are E. coli K12 strains derived from Dr. MartinRosenberg's laboratory at the National Institutes of Health.

3. Construction of the Vector Designed to Express the RecombinantProtein LPD-MAGE-3-His:

The rationale was to express MAGE 3 as a fusion protein using theN-terminal third of the lipidated protein D as fusion partner connectedat the N-terminus of MAGE-3 and a sequence of several histidine residues(His tail) placed at its C-terminus

Protein D is a lipoprotein (a 42 kDa immunoglobulin D binding proteinexposed on the surface of the Gram-negative bacterium Haemophilusinfluenzae). The protein is synthesized as a precursor with an 18 aminoacid residue signal sequence, containing a consensus sequence forbacterial lipoprotein (WO 91/18926).

When the signal sequence of a lipoprotein is processed during secretion,the Cys (at position 19 in the precursor molecule) becomes the aminoterminal residue and is concomitantly modified by covalent attachment ofboth ester-linked and amide-linked fatty acids.

The fatty acids linked to the amino-terminal cysteine residue thenfunction as membrane anchor.

The plasmid expressing the fusion protein was designed to express aprecursor protein containing the 18 amino acids signal sequence and thefirst 109 residues of the processed protein D, two unrelated amino acids(Met and Asp), amino acid residues 3 to 314 of MAGE-3, two Gly residuesfunctioning as a hinge region to expose the subsequent seven Hisresidues. (SEQ ID NO:2 encoded by SEQ ID NO:1)

The recombinant strain thus produces the processed lipidated His tailedfusion protein of 432 amino acid residues long (see FIG. 1), with theamino acids sequence described in SEQ ID NO:2 and the coding sequence isdescribed in SEQ ID NO:1.

4. Cloning Strategy for the Generation of the LPD-Mage-3-His FusionProtein (Vector pRIT14477):

A cDNA plasmid (from Dr Thierry Boon from the Ludwig Institute)containing the coding sequence for MAGE-3 gene (Gaugler B et al, 1994),and the vector PRIT 14586, containing the N terminal portion of theLipo-D-1/3 coding sequence (prepared as outlined in FIG. 2) were used.The cloning strategy included the following steps (FIG. 3).

a)—PCR amplification of the sequences presented in the plasmid cDNA MAGE3 using the oligonucleotide sense: 5′ gc gcc atg gat ctg gaa cag cgt agtcag cac tgc aag cct (SEQ ID NO:11), and the oligonucleotide antisense:5′ gcg tct aga tta atg gtg atg gtg atg gtg atg acc gcc ctc ttc ccc ctctct caa (SEQ ID NO:12); this amplification leads to the followingmodifications at the N terminus: changing of the first five codons to E.coli codon usage, replacement of the Pro codon by an Asp codon atposition 1, installation of an NcoI site at the 5′ extremity and finallyaddition of two 2 Gly codons and the 7 His codon followed by an XbaIsite at the C-terminus.

b)—Cloning into the TA cloning vector of invitrogen of the aboveamplified fragment and preparation of the intermediate vector pRIT14647.

c)—Excision of the NcoI XbaI fragment from plasmid pRIT14647 and cloninginto the vector pRIT 14586.

d)—Transformation of the host strain AR58.

e)—Selection and characterization of the E. coli strain transformantscontaining the plasmid pRIT 14477, expressing the LPD-MAGE-3-His fusionprotein.

Example II Preparation of the LPD1/3-MAGE-3-His Antigen 1. Growth andInduction of Bacterial Strain—Expression of LPD1/3-MAGE-3-His:

Cells of AR58 transformed with plasmid pRIT14477 were grown in 2 litreflasks, each containing 400 mL of LY12 medium supplemented with yeastextract (6.4 g/L) and kanamycin sulphate (50 mg/L). After incubation ona shaking table at 30° C. for 8+/−1 h, a small sample was removed fromeach flask for microscopic examination. The contents of the two flaskswere pooled to provide the inoculum for the 20 litre fermentor.

The inoculum (about 800 mL) was added to a pre-sterilised 20 litre(total volume) fermentor containing 7 litres of medium, supplementedwith 50 mg/L of kanamycin sulphate. The pH was adjusted to andmaintained at 6.8 by the periodic addition of NH₄OH (25% v/v), and thetemperature was adjusted to and maintained at 30° C. The aeration ratewas adjusted to and maintained at 12 litres of air/min and the dissolvedoxygen tension was maintained at 50% of saturation by feedback controlof the agitation speed. The over-pressure in the fermentor wasmaintained at 500 g/cm² (0.5 bar).

The fed-batch cultivation was carried out by controlled addition of acarbon feed solution. The feed solution was added at an initial rate of0.04 mL/min, and increased exponentially during the first 42 hours tomaintain a growth rate of 0.1 h⁻¹.

After 42 hours, the temperature in the fermentor was rapidly increasedto 39° C., and the feeding speed was maintained constant at 0.005 mL/gDCW/min during the induction phase for an additional 22-23 hours, duringwhich time intracellular expression of LPD-MAGE-3-His reached a maximumlevel.

Aliquots (15 mL) of broth were taken at regular intervals throughout thegrowth/induction phases and at the end of the fermentation to follow thekinetics of microbial growth and intracellular product expression and inaddition, to provide samples for microbial identification/purity tests.

At the end of fermentation, the optical density of the culture wasbetween 80 and 120 (corresponding to a cell concentration of between 48and 72 g DCW/L), and the total liquid volume was approximately 12litres. The culture was rapidly cooled to between 6 and 10° C., and thecells of ECK32 were separated from the culture broth by centrifugationat 5000×g at 4° C. for 30 minutes. The concentrated cells of ECK32 werequickly stored in plastic bags and immediately frozen at −80° C.

2. Extraction of the Protein:

The frozen concentrated cells of ECK32 were thawed to 4° C. before beingre-suspended in cell disruption buffer to a final optical density of 60(corresponding to a cell concentration of approximately 36 g DCW/L).

The cells were disrupted by two passes through a high-pressurehomogeniser (1000 bar). The broken cell suspension was centrifuged (×10000 g at 4° C. for 30 minutes) and the pellet fraction was washed twicewith Triton X100 (1% w/v)+EDTA (1 mM), followed by a wash with phosphatebuffered saline (PBS)+Tween 20 (0.1% v/v) and finally a wash with PBS.Between each washing stage, the suspension was centrifuged at ×10 000 gfor 30 minutes at 4° C., the supernatant was discarded and the pelletfraction was retained.

Example III Characterisation of Fusion Protein Lipo D—MAGE 3 1.Purification:

LPD-MAGE-3-His was purified from the cell homogenate using a sequence ofsteps described below:

-   -   a)—Solubilisation of the washed pellet fraction from cell        disruption,    -   b)—Chemical reduction of intra- and inter-protein disulphide        bonds followed by blocking of thiol groups to prevent oxidative        re-coupling,    -   c)—Microfiltration of the reaction mixture for the removal of        particulates and reduction of endotoxins,    -   d)—Capture and primary purification of LPD-MAGE-3-His by        exploitation of the affinity interaction between the        polyhistidine tail and zinc-loaded Chelating Sepharose,    -   e)—Removal of contaminant proteins by anion exchange        chromatography.

The purified LPD-MAGE 3-His was subjected to a number of polishingstages:

-   -   f)—Buffer exchange/urea removal by size exclusion chromatography        using Superdex 75,    -   g)—In-process filtration,    -   h)—Buffer exchange/desalting by size exclusion chromatography        using Sephadex G25.

Each of these steps is described in more detail below:

1.1)—Solubilisation of Cell Homogenate Pellet

The pellet fraction from the final washing stage (as described above)was re-solubilised overnight in 800 mL of a solution of guanidinehydrochloride (6M) and sodium phosphate (0.1 M, pH 7.0) at 4° C.

1.2)—Reduction and Carboxymethylation

The solubilised material (a pale yellow, turbid suspension) was flushedwith argon to purge any remaining oxygen, and a stock solution of2-mercaptoethanol (14M) was added to provide a final concentration of4.3M (which corresponded to 0.44 mL of 2-mercaptoethanol per mL ofsolution).

The resulting solution was divided and transferred into two glass flaskswhich were both heated to 95° C. in a water bath. After 15 minutes at95° C., the flasks were removed from the water bath and allowed to cool,whereupon the contents were pooled into a foil-covered beaker (5 L),placed on ice, and solid iodoacetamide added with vigorous mixing toprovide a final concentration of 6M (which corresponded to 1.11 g ofiodoacetamide per mL of solution). The mixture was held on ice in thedark for 1 hour to ensure complete solubilisation of iodoacetamide,before being neutralised (maintaining vigorous mixing and continuous pHmonitoring) by the addition of approximately 1 litre of sodium hydroxide(5 M) to give a final pH of 7.5-7.8.

The resulting mixture was maintained on ice in the dark for a further 30minutes, after which time the pH was re-adjusted to pH 7.5-7.8.

1.3)—Microfiltration

The mixture was microfiltered in an Amicon Proflux M12 tangential-flowunit equipped with a Minikros hollow fibre cartridge (ref No.M22M-600-01N; area 5,600 cm², 0.2 μm). The permeate was retained forsubsequent chromatographic purification.

1.4)—Metal (Zn²⁺) Chelate Chromatography (IMAC)

Metal chelate chromatography was performed with Chelating Sepharose FF(Pharmacia Biotechnology Cat. No. 17-0575-01) packed into a BPG 100/500column (Pharmacia Biotechnology Cat No. 18-1103-01). The dimensions ofthe packed bed were: diameter 10 cm; cross-sectional area 79 cm²; bedheight 19 cm; packed volume 1,500 mL. The empty column was sanitisedwith sodium hydroxide (0.5M), then washed with purified water.

The support (delivered in 20% v/v ethanol) was washed with purifiedwater (8 litres) on a Buchner funnel (under vacuum) and charged withzinc by passing at least 15 litres of a solution of ZnCl₂ (0.1M). Excesszinc was removed by washing the support with 10 litres of purifiedwater, until the pH of the outlet liquid reached the pH of the ZnCl₂solution (pH 5.0). The support was then equilibrated with 4 litres of asolution containing guanidine hydrochloride (6M) and sodium phosphate(0.1M, pH 7.0).

The permeate from microfiltration, containing LPD-MAGE-3-His, was mixedwith the support (batch binding), before loading and packing the BPGcolumn with the solution containing guanidine hydrochloride (6M) andsodium phosphate (0.1M, pH 7.0).

The next stages of metal chelate chromatography were conducted at aneluent flow rate of 60 mL/min. The column was washed, first with thesolution containing guanidine hydrochloride (6M) and sodium phosphate(0.1M, pH 7.0), then with the solution containing urea (6M) and sodiumphosphate (0.1M, pH 7.0), until the column eluent attained zeroabsorbance at OD₂₈₀ nm (baseline).

The semi-pure LPD-MAGE-3-His protein fraction was eluted with 2 columnvolumes of a solution containing urea (6M), sodium phosphate (0.1M, pH7.0) and imidazole (0.5M). The conductance of this fraction wasapproximately 16 mS/cm.

1.5)—Anion Exchange Chromatography

Before continuing with anion exchange chromatography, the conductance ofthe semi-pure LPD-MAGE-3-His protein fraction was reduced toapproximately 4 mS/cm by dilution with a solution containing urea (6M)and Tris-HCl (20 mM, pH 8.0).

Anion exchange chromatography was performed using Q-Sepharose FF(Pharmacia Biotechnology, Cat. No. 17-0510-01) packed in a BPG 200/500column (Pharmacia Biotechnology Cat. No. 18-1103-11). The dimensions ofthe packed bed were: diameter 10 cm; cross-sectional area 314 cm²; bedheight 9 cm; packed volume 2,900 mL.

The column was packed (with 20% v/v ethanol) and washed with 9 litres ofpurified water at an eluent flow rate of 70 mL/min. The packed columnwas sanitised with 3 litres of sodium hydroxide (0.5M), washed with 30litres of purified water, then equilibrated with 6 litres of a solutioncontaining urea (6M) and Tris-HCl (20 mM, pH 8.0). The diluted,semi-purified LPD-MAGE-3-His was loaded onto the column and then washedwith 9 litres of a solution containing urea (6M), Tris-HCl (20 mM, pH8.0), EDTA (1 mM) and Tween (0.1%), until the absorbance (280 nm) of theeluent fell to zero.

A further washing step was performed with 6 litres of a solutioncontaining urea (6M) and Tris-HCl (20 mM, pH 8.0).

The purified LPD-MAGE-3-His was eluted from the column with a solutioncontaining urea (6M), Tris-HCl (20 mM, pH 8.0) and NaCl (0.25M).

1.6)—Size Exclusion Chromatography

The removal of urea from purified LPD-MAGE-3-His and the buffer exchangewere both achieved by size exclusion chromatography. This was performedusing Superdex 75 (Pharmacia Biotechnology Cat. No. 17-1044-01) packedin an XK 50/100 column (Pharmacia Biotechnology Cat. No. 18-8753-01).The dimensions of the packed bed were: diameter 5 cm; cross-sectionalarea 19.6 cm²; bed height 90 cm; packed volume 1,800 mL.

The column was packed in ethanol (20%) and washed with 5 litres ofpurified water at an effluent flow rate of 20 mL/min. The column wassanitised with 2 litres of sodium hydroxide (0.5M), washed with 5 litresof purified water, then equilibrated with 5 litres of phosphate-bufferedsaline containing Tween 80 (0.1% v/v).

The purified LPD-MAGE-3-His fraction (maximum 500 mL/desalting run) wasloaded onto the column at an eluent flow rate of 20 mL/min. The desaltedpurified LPD-MAGE-3-His was eluted from the column with 3 litres of PBScontaining Tween 80 (0.1% v/v).

The fraction containing LPD-MAGE-3-His eluted at the void volume of thecolumn.

1.7)—In-Process Filtration

The bulk LPD-MAGE-3-His from size exclusion chromatography was filteredthrough a 0.22 μm membrane in a laminar flow hood (class 10.000). Thefiltered bulk was frozen at −80° C. and stored until the desalting step.

1.8)—Desalting Chromatography

Since the osmolality of the final bulk should be less than 400 mOsM, afurther buffer exchange step was required to reduce the saltconcentration. This was performed by a desalting chromatographic stepusing Sephadex G25 (Pharmacia Biotechnology Cat. No. 17-0033-02) packedin a BPG 100/950 column (Pharmacia Biotechnology Cat. No. 18-1103-03).The dimensions of the packed bed were: diameter 10 cm; cross-sectionalarea 78.6 cm²; bed height 85 cm; packed volume 6,500 mL.

The Sephadex G25 was hydrated with 7 litres of purified water andallowed to swell overnight at 4° C. The gel was then packed in thecolumn with pure water at an eluent flow rate of 100 mL/min.

The column was sanitised with 6 litres of sodium hydroxide (0.5M), thenequilibrated with 10 litres of a solution containing sodium phosphate(10 mM, pH 6.8), NaCl (20 mM) and Tween 80 (0.1% v/v).

The purified LPD-MAGE-3-His fraction (maximum 1500 mL/desalting run) wasloaded onto the column at an eluent flow rate of 100 mL/min. Thedesalted purified LPD-MAGE-3-His fraction eluted at the void volume ofthe column, was sterile filtered through a 0.22 μm membrane and storedat −80° C.

The final bulk protein is thawed to +4° C. before being aliquoted intovials and freeze-dried in a lactose excipient (3.2%).

2. Analysis on Coomassie-Stained SDS-Polyacrylamide Gels:

The LPD-MAGE-3-His purified antigen was analysed by SDS-PAGE on a 12.5%acrylamide gel in reducing conditions.

The protein load was 50 μg for Coomassie blue staining and 5 μg forsilver nitrate staining Clinical lot 96K19 and pilot lot 96J22 wereanalyzed. One major band corresponding to a molecular weight of 60 kDawas visualised. Two minor additional bands of approximately 45 kDa and35 kDa were also seen.

3. Western Blot Analysis:

The peptides revealed by SDS-PAGE analysis of the LPD-MAGE-3-His proteinwere identified by Western blot using mouse monoclonal antibodies. Theseantibodies were developed in-house using a purified preparation of theMAGE-3-His protein (this protein does not contain the LPD part of theLPD-MAGE-3-His).

Two monoclonal antibody preparations (Mab 22 and Mab 54) have beenselected on the basis of their suitability for Western blot analysis andused in the identity test for lot release. FIG. 4 shows the bandpatterns obtained for lots 96K19 and 96J22 after staining with Mabs 32and 54. Six hundred (600) ng of protein were resolved on a 12.5%SDS-PAGE, transferred to a nylon membrane, reacted with Mabs 32 and 54(60 μg/ml) and revealed with anti-mouse antibodies coupled toperoxidase.

The 60 kDa and 30 kDa peptide detected by SDS-PAGE are revealed by bothMabs.

Example IV 1. Vaccine Preparation Using LPD-MAGE-3-His Protein

The vaccine used in these experiments is produced from a recombinantDNA, encoding a Lipoprotein D 1/3-MAGE-3-His, expressed in E. coli fromthe strain AR58, either adjuvanted or not. As an adjuvant, theformulation comprises a mixture of 3 de —O-acylated monophosphoryl lipidA (3D-MPL) and QS21 in an oil/water emulsion. The adjuvant system SBAS2has been previously described WO 95/17210.

3D-MPL: is an immunostimulant derived from the lipopolysaccharide (LPS)of the Gram-negative bacterium Salmonella minnesota. MPL has beendeacylated and is lacking a phosphate group on the lipid A moiety. Thischemical treatment dramatically reduces toxicity while preserving theimmunostimulant properties (Ribi, 1986). Ribi Immunochemistry producesand supplies MPL to SB-Biologicals.

Experiments performed at Smith Kline Beecham Biologicals have shown that3D-MPL combined with various vehicles strongly enhances both the humoraland a TH1 type of cellular immunity.

QS21: is a natural saponin molecule extracted from the bark of the SouthAmerican tree Quillaja saponaria Molina. A purification techniquedeveloped to separate the individual saponines from the crude extractsof the bark, permitted the isolation of the particular saponin, QS21,which is a triterpene glycoside demonstrating stronger adjuvant activityand lower toxicity as compared with the parent component. QS21 has beenshown to activate MHC class I restricted CTLs to several subunit Ags, aswell as to stimulate Ag specific lymphocytic proliferation (Kensil,1992). Aquila (formally Cambridge Biotech Corporation) produces andsupplies QS21 to SB-Biologicals.

Experiments performed at SmithKline Beecham Biologicals havedemonstrated a clear synergistic effect of combinations of MPL and QS21in the induction of both humoral and TH1 type cellular immune responses.

The oil/water emulsion is composed of an organic phase made of 2 oils (atocopherol and squalene), and an aqueous phase of PBS containing Tween80 as emulsifier. The emulsion comprised 5% squalene 5% tocopherol 0.4%Tween 80 and had an average particle size of 180 nm and is known as SB62(see WO 95/17210).

Experiments performed at SmithKline Beecham Biologicals have proven thatthe adjunction of this O/W emulsion to 3D-MPL/QS21 (SBAS2) furtherincreases the immunostimulant properties of the latter against varioussubunit antigens.

2. Preparation of Emulsion SB62 (2 Fold Concentrate)

Tween 80 is dissolved in phosphate buffered saline (PBS) to give a 2%solution in the PBS. To provide 100 ml two fold concentrate emulsion 5 gof DL alpha tocopherol and 5 ml of squalene are vortexed to mixthoroughly. 90 ml of PBS/Tween solution is added and mixed thoroughly.The resulting emulsion is then passed through a syringe and finallymicrofluidised by using an M110S microfluidics machine. The resultingoil droplets have a size of approximately 180 nm.

3. Preparation of Lipoprot. D1/3-MAGE-3-His QS21/3D MPL Oil in Water(SBAS2) Formulation

The adjuvant is formulated as a combination of MPL and QS21, in anoil/water emulsion. This preparation is delivered in vials of 0.7 ml tobe admixed with the lyophilised antigen (vials containing from 30 to 300μg antigen).

The composition of the adjuvant diluent for the lyophilised vaccine isas follows:

Quantity Ingredients: (per dose): Adjuvants SB62 Emulsion: 250 μlSqualene 10.7 mg DL α-tocopherol 11.9 mg Tween 80 4.8 mg MonophosphorylLipid A 100 μg QS21 100 μg Preservative Thiomersal 25 μg Buffer Waterfor injection q.s. ad 0.5 ml Dibasic sodium phosphate 575 μg Monobasicpotassium phosphate 100 μg Potassium chloride 100 μg Sodium chloride 4.0mg

The final vaccine is obtained after reconstitution of the lyophilisedLPD-MAGE-3-His preparation with the adjuvant or with PBS alone.

The adjuvants controls without antigen were prepared by replacing theprotein by PBS.

4. Vaccine Antigen: Fusion Protein Lipoprotein D1/3-MAGE-3-His

Lipoprotein D is a lipoprotein exposed on the surface of theGram-negative bacteria Haemophilus influenzae.

The inclusion of the first 109 residues of the processed protein D asfusion partner is incorporated to provide the vaccine antigen with aT-cell epitopes. Besides the LPD moiety, the protein contains twounrelated amino acids (Met and Asp), amino acid residues 3-314 ofMage-3, two Gly residues functioning as hinge region to expose thesubsequent seven His residues.

Example V Immunogenicity of LPD-MAGE-3-His in Mice and Monkeys

In order to test the antigenicity and immunogenicity of the human MAGE-3protein, the candidate vaccine was injected into 2 different mousestrains (C57BL/6 and Balb/C), varying in their genetic background andMHC alleles. For both mouse strains, potential MHC class-I and MHCclass-II peptide motifs were theoretically predicted for the MAGE partof the LPD-MAGE-3-His fusion protein.

a)—Immunization Protocol:

5 mice of each strain were injected twice at 2 weeks interval in thefoot pad with 5 μg of LPD-MAGE-3-His, formulated or not in SBAS2 at1/10th of the concentration used in human settings.

b)—Proliferation Assay:

Lymphocytes were prepared by crushing the spleen or the popliteal lymphnodes from the mice, 2 weeks after the last injection. 2×10⁵ cells wereplaced in triplicate in 96 well plates and the cells were re-stimulatedin vitro for 72 hours with different concentrations (1-0.1 μg/ml) ofHis-Mage 3 as such or coated onto latex micro-beads.

An increased MAGE-3 specific lymphoproliferative activity was observedwith both spleen cells (see FIGS. 5 and 7) and lymph node cells (seeFIGS. 6 and 8) from either C57BL/6 or Balb/C mice injected with theLPD-MAGE-3-His protein, as compared with the lymphoproliferativeresponse of mice having received the SBAS-2 formulation alone or PBS.

Moreover, a significant higher proliferative response was obtained withlymphocytes from mice immunized with LPD-MAGE-3-His in the adjuvantSBAS2 (see FIGS. 6 and 8).

c)—Conclusion:

LPD-MAGE-3-His is immunogenic in mice, and this immunogenicity can beincreased by the use of the SBAS2 adjuvant formulation.

2. Antibody Response:

a)—Immunization Protocol:

Balb/c or C57BL/6 mice were immunized by 2 intra foot pad injections at2 weeks interval with either PBS, or SBAS2, or 5 μG of LPD-MAGE-3-His,or 5 μG of LPD-MAGE-3-His+SBAS2.

Three and five animals were used in the control groups and in the testedgroups respectively.

b)—Indirect ELISA:

Two weeks after the second injection, individual sera were taken andsubmitted to an indirect ELISA.

2 μG/ml of purified His MAGE 3 was used as coated antigen. Aftersaturation during 1 hour at 37° C., in PBS+1% newborn calf serum, thesera were serially diluted (starting at 1/1000) in the saturation bufferand incubated overnight at 4° C., or 90 minutes at 37° C. After washingin PBS/Tween 20.01%, Biotinylated goat anti-mouse total IgG ( 1/1000) orgoat anti-mouse IgG1, IgG2a, IgG2b antisera ( 1/5000) were used assecond antibodies. After 90 minutes incubation at 37° C. Streptavidincoupled to peroxidase was added, and TMB (tetra-methyl-benzidineperoxide) was used as substrate. After 10 minutes the reaction wasblocked by addition of H₂SO₄ 0.5M, and the O.D. was determined

c)—Results:

FIG. 9 compares between the different groups of mice (N=5/group), therelative mean midpoint titer of the sera, which consists in the meandilution needed to reach the midpoint of the curves.

These results show that in both mouse strains tested, a weak Ab responseis mounted after 2 injections of LPD-MAGE-3-His alone, but that higheranti-MAGE 3 Ab concentrations are generated when LPD-MAGE-3-His isinjected in the presence of SBAS2. Thus, only 2 injections ofLPD-MAGE-3-His+SBAS2, at 2 weeks interval, are sufficient to generatethe high Ab response observed.

The better Ab response observed in the Balb/c mice as compared with theresponse obtained in the C57BL/6 mice can be explained by differences inhaplotypes or in background between these 2 strains, even though the Abtitre achieved in C57BL/6 mice is also higher after injections ofLPD-MAGE-3-His+SBAS2 than after injections with LPD-MAGE-3-His alone.

The Ig subclasses-specific anti-MAGE-3 responses after vaccinations inthe different groups of mice can be seen on the FIGS. 10 and 11, whichgive a comparison of the mean midpoint dilution of the sera.

Neither IgA, nor IgM were detected in any of the serum samples even fromthe mice vaccinated with LPD-MAGE-3-His in the adjuvant SBAS2.

On the contrary, the total IgG level was slightly higher in the serafrom mice vaccinated with LPD-MAGE-3-His alone, and significantlyincreased in the sera of animals injected with LPD-MAGE-3-His in SBAS2.

The analysis of the different IgG-subclasses concentrations show that amixed Ab response was induced in the mice, since the levels of all IgGsubclasses tested (IgG1, IgG2a, IgG2b) were higher in mice vaccinatedwith the adjuvanted Ag than in mice injected with the Ag or the adjuvantalone.

The nature of this mixed Ab response after vaccination with LipoD-MAGE 3in the presence of SBAS2 seems however to depend on the mouse strain,since IgG1 and IgG2b were predominantly found in the sera of Balb/c andC57BL/6 mice respectively.

3. Immunogenicity of Lipoprotein D1/3 MAGE-3-His+SBAS2 Adjuvant inRhesus Monkeys

Three groups of five Rhesus (Macaca Mulatta) animals were selected.RTS,S and gp120 were used as positive control.

Groups:

Group 1

right leg: RTS,S/SBAS2

left leg: GP120/SBAS2

Group 2

right leg: RTS,S/SB26T

left leg: GP120/SB26T

Group 3

right leg: LipoD1/3 Mage 3 His/SBAS2

The animals received vaccine at day 0 and were boosted at day 28, and 84and bled to determine their antibody response to both the MAGE 3 andprotein D component. The vaccines were administered intramuscularly as abolus injection (0.5 ml) in the posterior part of the right leg.

Small blood samples were taken every 14 days. Unheparinized bloodsamples of 3 ml were collected from the femoral vein, were allowed toclot for at least 1 hour and centrifuged at room temperature for 10minutes at 2500 rpm.

Serum was removed, frozen at −20° C. and sent for determination of theantibody levels by specific Elisa.

96 well microplates (maxisorb Nunc) were either coated with 5 μg of HisMage 3 or Protein D overnight at 4° C. After 1 hour saturation at 37° C.with PBS NCS 1%, serial dilution of the rabbit sera were added for 1 H30 at 37° C. (starting at 1/10), after 3 washings in PBS Tween, antirabbit biotinylated serum (Amersham ref RPN 1004 lot 88) was added (1/5000). Plates were washed and peroxydase couple streptavidin ( 1/5000)was added for 30 minutes at 37° C. After washing, 50 μl TMB (BioRad) wasadded for 7 minutes and the reaction was stopped with H2S04 0.2M, OD wasmeasured at 450 nm. Midpoint dilutions were calculated by SoftmaxPro.

Antibody Response:

Small blood samples were taken every 14 days to follow the kinetic ofthe antibody response to Mage 3 by ELISA. The results indicates thatafter one injection of LPD1/3 Mage 3 His+SBAS2, the Mage 3 specifictotal Ig titer was low, a clear boost was seen in 3 out of 5 animalsafter a second and a third injection of LipoD1/3 Mage 3+adjuvant in thesame monkeys. The poor responders remained negative even after 3injections. 28 days post II or post III, the antibody titers hasreturned to basal levels. The subclass of these antibodies wasdetermined as predominantly IgG and not IgM. The switch to IgG suggeststhat a T helper response has been triggered. The Protein D specificantibody response, although weaker, is exactly parallel to the Mage 3antibody response.

Example VI 1. LPD—MAGE 1 His

In an analogous fashion—LPD—MAGE 1-His was prepared. The amino acid andDNA sequences are depicted in SEQ ID NO:3 and SEQ ID NO:4. The resultingprotein was purified in an analogous manner to the LPD-MAGE-3-Hisprotein. Briefly, the cell culture were homogenated and treated with 4Mguanidine HCl and 0.5 M beta mercaptoethanol in the presence of 0.5%Empigen detergent. The product was filtered and the permeate treatedwith 0.6 M iodoacetamide. The carboxyamidated fractions was subjected toIMAC (zinc Chealate-sepharose FF) chromatography. The column was firstequilbrated and washed with a solution containing 4M guanidine. HCl andsodium phosphate (20 mM, pH7.5) and 0.5% Empigen, then the column waswashed with a solution containing 4M urea in sodium phosphate (20 mM,pH7.5) 0.5% Empigen buffer. The protein was eluted in the same buffer,but with increasing concentration of Imidazole (20 mM, 400 mM and 500mM).

The eluate was diluted with 4M Urea. The Q-sepharose column wasequilibrated and washed with 4M Urea in 20 mM phosphate buffer (pH7.5)in the presence of 0.5% Empigen. A second wash was performed in the samebuffer, but devoid of the detergent. The protein eluted in the samebuffer but with increasing Imidazole (150 mM, 400 mM, 1M). The eluatewas ultra filtered.

Example VII Construction of the Expression Plasmid pRIT14426 andTransformation of the Host Strain AR58 to Produce NS1-MAGE-3 His ProteinDesign:

The design of the fusion protein NS1,-MAGE-3-His to be expressed in E.coli is described in FIG. 12.

The primary structure of the resulting protein has the sequence setforth in SEQ ID No:5.

The coding sequence (SEQ ID No:6) corresponding to the above proteindesign was placed under the control of λpL promoter in a E. coliexpression plasmid.

The Cloning Strategy for the Generation of NS₁-MAGE-3-His FusionProtein:

The starting material was a cDNA plasmid received from Dr Tierry Boonfrom the Ludwig Institute, containing the coding sequence for MAGE-3gene and the vector PMG81, containing the 81aa of NS₁ (Non structuralprotein) coding region from Influenza.

The cloning strategy outlined in FIG. 13 included the following steps:

a) PCR amplification of the sequences presented in the plasmid cDNAMAGE-3 using the oligonucleotide sense: 5′ gc gcc atg gat ctg gaa cagcgt agt cag cac tgc aag cct (SEQ ID NO:11), and the oligonucleotideantisense: 5′ gcg tct aga tta atg gtg atg gtg atg gtg atg acc gcc ctcttc ccc ctc tct caa (SEQ ID NO:12).

This amplification leads to the following modifications at the Nterminus: changing of the first five codons to the E. coli codon usage,replacement of the Pro codon by an Asp codon at position 1, installationof an NcoI site at the 5′ extremity and finally addition of the 2 Glycodons and the 7 His codon followed by an XbaI site at the C-terminus.

b) Cloning into the TA cloning vector of invitrogen of the aboveamplified fragment and preparation of the intermediate vector pRIT14647

c) Excision of the NcoI XbaI fragment form plasmid pRIT14647 and cloninginto the vector pRIT PMG81

d) Transformation of the host strain AR58

e) Selection and characterization of the E. coli strain transformantscontaining the plasmid pRIT14426 (see FIG. 14) expressing theNS1-MAGE-3-His fusion protein

Characterization of the Recombinant NS₁-MAGE-3-His (pRIT14426):

Bacteria were grown on LB Medium supplemented with 50 μg/ml kanamycin at30° C. When the culture had reached OD=0.3 (at 620 nm), heat inductionwas achieved by raising the temperature to 42° C.

After 4 hours induction, cells were harvested, resuspended in PBS andlysed (by disintegration) by pressing three times in the French press.After centrifugation (60 minutes at 100,000 g), pellet supernatant andtotal extract were analyzed by SDS-PAGE. Proteins were visualized inCoomassie B1 stained gels where the fusion protein represented about 1%of the total E. coli proteins. The recombinant protein appeared as asingle band with an apparent MW of 44.9 K. The fusion protein wasidentified by Western Blot analysis using anti-NS 1 monoclonal.

Example VIII Purification of NS1-MAGE 3-His (E. coli) for Rabbit/MiceImmunization

Purification Scheme:

The following purification scheme was used to purify the antigen:

a. Lysis

Bacterial cells (23 g) were lysed in 203 ml of a 50 mM PO₄ pH7 buffer byRannie (homogeniser) and the lysate was centrifuged in a JA 20 rotor at15,000 rpm during 30 minutes.

The supernatant was discarded.

b. Antigen Solubilisation

⅓ of the pellet was resolubilised 0/N at 4° C. in 34 ml of 100 mM PO₄—6M GuHCl pH7. After centrifugation in a JA 20 rotor at 15,000 rpm for 30minutes, the pellet was discarded and the supernatant was furtherpurified by IMAC.

c. Affinity Chromatography: Ni²+-NTA Agarose (Qiagen)

Column volume: 15 ml (16 mm×7.5 cm)

Packing buffer: 0.1 M PO₄—6 M GuHCl pH7

Sample buffer: idem

Washing buffer: 0.1 M PO₄—6 M GuHCl pH7

-   -   0.1M PO₄—6 M urea pH7

Elution: imidazol gradient (0→250 mM) in 0.1 M PO₄ buffer pH7supplemented with 6 M urea.

Flow rate: 2 ml/min

a. Concentration:

Antigen positive fractions of the IMAC eluate (160 ml) were pooled andconcentrated to 5 ml in an Amicon stirred cell on a Filtron membrane(type Omega cut-off 10,000). The purity at this stage is about 70% asestimated by SDS-PAGE.

b. Preparative Electrophoresis (Prep Cell Biorad)

2.4 ml of the concentrated sample was boiled in 0.8 ml reducing samplebuffer and loaded on a 10% acrylamide gel. The antigen was eluted in aTris-Glycine buffer pH 8.3 supplemented with 4% SDS and Ns₁—MAGE 3 Hispositive fractions were pooled.

a. TCA Precipitation:

The antigen was TCA precipitated and after centrifugation in a JA 20rotor at 15,000 rpm for 20 minutes, the supernatant was discarded. Thepellet was resolubilised in PBS buffer pH 7.4.

The protein is soluble in PBS after freeze/thaw does not show anydegradation when stored for 3 hours at 37° C. and has an apparentmolecular weight of approximately 50,000 Daltons as determined by SDS(12.5% PAGE).

Example IX Preparation of the E. coli Strain Expressing a Fusion ProteinCLYTA-MAGE-1-His Tail

1. Construction of the Expression Plasmid pRIT14613 and Transformationof the Host Strain AR58:

Protein Design:

The design of the fusion protein Clyta-Mage-1-His to be expressed in E.coli is described in FIG. 15.

The primary structure of the resulting protein has the sequence setforth in SEQ ID No:7.

The coding sequence (see SEQ ID No:8) corresponding to the above proteindesign was placed under the control of λ pL promoter in a E. coliexpression plasmid.

Cloning:

The starting material was the vector PCUZ1 that contains the 117C-terminal codons of the LytA coding region from Streptococcuspneumoniae and the vector pRIT14518, in which we have previouslysubcloned the MAGE-1 gene cDNA from a plasmid received from Dr ThierryBoon from the Ludwig Institute.

The cloning strategy for the expression of CLYTA-Mage-1-His protein (seeoutline in FIG. 16) included the following steps:

2. Preparation of the CLYTA-Mage-1-his Coding Sequence Module:

a) The first step was a PCR amplification, destined to flank the CLYTAsequences with the NdeI-AflIII restriction sites. The PCR amplificationwas done using the plasmid PCUZ1 as template and as primers theoligonucleotide sense: 5′ tta aac cac acc tta agg agg ata taa cat atgaaa ggg gga att gta cat tca gac (SEQ ID NO:13), and the oligonucleotideantisense: 5′ GCC AGA CAT GTC CAA TTC TGG CCT GTC TGC CAG (SEQ IDNO:14). This leads to the amplification of a 378 nucleotides long CLYTAsequence.

b) The second step was linking of CLYTA sequences to the MAGE-1-Hissequences, to generate the coding sequence for the fusion protein. Thisstep included the excision of a NdeI-AflIII Clyta fragment and insertioninto the vector pRIT14518 previously opened by NdeI and NcoI (NcoI andAflIII compatible) restriction enzymes and gave rise to the plasmidpRIT14613.

c) Transformation of the host strain AR58

d) Selection and characterization of the E. coli transformant (KANresistant) containing the plasmid pRIT14613. (See FIG. 16)

1. Characterization of the Recombinant Protein CLYTA-MAGE-1-His(pRIT14613):

Bacteria were grown on LB Medium supplemented with 50 μg/ml kanamycin at30° C. When the culture had reached OD=0.3 (at 620 nm), heat inductionwas achieved by raising the temperature to 38° C.

After 4 hours induction, cells were harvested, resuspended in PBS andlysed (by disintegration) by one shot. After centrifugation, pelletsupernatant and total extract were analyzed by SDS-PAGE. Proteins werevisualized in Coomassie B1 stained gels, where the fusion proteinrepresented about 1% of the total E. coli proteins. The recombinantprotein appeared as a single band with an apparent MW of about 49 kD.The fusion protein was identified by Western Blot analysis usinganti-Mage-1 polyclonal antibodies.

Reconstitution of the Expression Unit Composed by the Long λ pL Promoter(Useful for Nalidixic Acid Induction) and the CLYTA-Mage-1 CodingSequence pRIT14614):

A EcoRI—NCO₁ restriction fragment containing the long PL promoter and apart of CLYTA sequences was prepared from plasmid pRIT DVA6 and insertedbetween the EcoRI—NCO₁ sites of plasmid pRIT14613.

The recombinant plasmid pRIT14614 was obtained.

The recombinant plasmid pRIT14614 (see FIG. 17) encoding the fusionprotein CLYTA-Mage-1-His was used to transform E. coli AR120. A Kanresistant candidate strain was selected and characterized.

Characterization of the Recombinant Protein:

Bacteria were grown on LB Medium supplemented with 50 mg/ml kanamycin at30° C. When the culture had reached OD=400 (at 620 nm) Nalidixic acidwas added to a final concentration of 60 mg/ml.

After 4 hours induction, cells were harvested, resuspended in PBS andlysed by desintegration (disintegration CLS “one shot” type). Aftercentrifugation, pellet supernatant and total extract were analyzed bySDS-PAGE. Proteins were visualized in Coomassie Bleu stained gels, wherethe fusion protein represented about 1% of the total E. coli proteins.The fusion protein was identified by Western blot analysis using rabbitsanti-Mage-1 polyclonal antibodies. The recombinant protein appeared as asingle band with an apparent MW of about 49 kD.

Example X CLYTA-MAGE-3-HIS

A: Tumour rejection recombinant antigen: a fusion proteinCLYTA-Mage-3-His where the C-lyt A fusion partner lead to expression ofa soluble protein, act as affinity tag and provides a useful T-helper.

Preparation of the E. coli strain expressing a fusion proteinCLYTA-Mage-3-His tail

Construction of the expression plasmid pRIT14646 and transformation ofthe host strain AR 120:

Protein Design:

The design of the fusion protein Clyta-Mage-3-His to be expressed in E.coli is described in FIG. 18.

The primary structure of the resulting protein has the sequencedescribed in SEQ ID No:9 and the coding sequence in SEQ ID No:10.

The coding sequence corresponding to the above protein design was placedunder the control of λ pL promoter in a E. coli expression plasmid.

Cloning:

The starting material was the vector PCUZ1 that contains the 117C-terminal codons of the LytA coding region from Streptococcuspneumoniae, described in Gene 43, (1986) p. 265-272 and the vectorpRIT14426, in which we have previously subcloned the MAGE-3 gene cDNAfrom a plasmid received from Dr Tierry Boon from the Ludwig Institute.

The cloning strategy for the expression of CLYTA-MAGE-3-His protein (seeoutline in FIG. 19) included the following steps:

1—Preparation of the CLYTA-MAGE-3-His Coding Sequence Module:

1.1 The first step was a PCR amplification, destined to flank the CLYTAsequences with the AflII and AflIII restriction sites. The PCRamplification was done using the plasmid PCUZ1 as template and asprimers the oligonucleotide sense: 5′ tta aac cac acc tta agg agg atataa cat atg aaa ggg gga att gta cat tca gac (SEQ ID NO:13), and theoligonucleotide antisense: 5′ ccc aca tgt cca gac tgc tgg cca att ctggcc tgt ctg cca gtg (SEQ ID NO:15). This leads to the amplification of a427 nucleotides long CLYTA sequence. The above amplified fragment wascloned into the TA cloning vector of Invitrogen to get the intermediatevector pRIT14661

1.2 The second step was linking of CLYTA sequences to the MAGE-3-Hissequences, to generate the coding sequence for the fusion protein. Thisstep included the excision of a AflII-AflIII Clyta fragment andinsertion into the vector pRIT14426 previously opened by Afl II and NcoI(NcoI and AflII compatible) restriction enzymes and gave rise to theplasmid pRIT14662.

2.—Reconstitution of the Expression Unit Composed by the Long λ pLPromoter (Useful for Nalidixic Acid Induction) and the CLYTA-Mage-3Coding Sequence:

A BglII-XbaI restriction fragment containing the short pL promoter andthe CLYTA-Mage-3-His coding sequences was prepared from plasmidpRIT14662. and inserted between the BglII-XbaI sites of plasmid TCM67 (apBR322 derivative containing the resistance to ampicillin, and the longλ pL promoter, described in the international applicationPCT/EP92/01827). The plasmid pRIT14607 was obtained.

The recombinant plasmid pRIT14607 encoding the fusion proteinClyta-Mage-3 His was used to transform E. coli AR 120 (Mott et a1.1985,Proc. Natl. Acad. Sci, 82: 88). An ampicillin resistant candidate strainwas selected and characterized.

3. Preparation of Plasmid pRIT 14646:

Finally a plasmid similar to pRIT 14607 but having the Kanamycinselection was constructed (pRIT 14646)

Characterization of the Recombinant Protein:

Bacteria were grown on LB Medium supplemented with 50 mg/ml kanamycin at30° C. When the culture had reached OD=400 (at 600 nm) Nalidixic acidwas added to a final concentration of 60 ?g/ml.

After 4 hours induction, cells were harvested, resuspended in PBS andlysed by desintegration (desintegration CLS “one shot” type). Aftercentrifugation, pellet supernatant and total extract were analyzed bySDS-PAGE. Proteins were visualized in Coomassie Bleu stained gels, wherethe fusion protein represented about 1% of the total E. coli proteins.The fusion protein was identified by Western blot analysis using rabbitsanti-Mage-3 polyclonal antibodies. The recombinant protein appeared as asingle band with an apparent MW of about 58 kD.

Example XI Purification of the Recombinant Protein CLYTA-Mage-3 His

The recombinant bacteria AR120 (pRIT 14646) were grown in a 20 Littersfermentor under fed-batch conditions at 30°. The expression of therecombinant protein was induced by adding Nalidixic acid at a finalconcentration of 60 ?g/ml. Cells were harvested at the end offermentationand and lyzed at 600D/600 by two passages through a FrenchPress disrupter (20 000 psi). Lysed cells were pelleted 20 min at 15 000g at 4° C. Supernatant containing the recombinant protein was loadedonto exchange DEAE Sepharose CL6B resin (Pharmacia) pre-equilibrated in0.3M NaCl, 20 mM Tris HCl pH 7.6 Buffer A. After a column wash withbuffer A, fusion protein was eluted by 2% choline in (Buffer A).Positive antigen fractions, as revealed by Western blotting analysisusing an anti Mage-3 antibody, were pooled. DEAE-eluted antigen wasbrought to 0.5% Empigen BB (a zwitterionic detergent) and to 0.5 M NaClbefore loading onto an Ion Metal Affinity chromatography columnpreequilibrated in 0.5% Empigen BB, 0.5 M NaCl, 50 mM phosphate bufferpH 7.6 (Buffer B).

IMAC column was washed with buffer B until 280 nm absorbency reached thebase line. A second wash in buffer B without Empigen BB (Buffer C) inorder to eliminate the detergent was executed before Antigen elution byan Imidazole gradient 0-250 mM Imidazole in buffer C.

0.090-0.250 M Imidazole fractions were pooled, concentrated on a 10 kDaFiltron omega membrane before dialysis versus PBS buffer.

CONCLUSION

We have demonstrated that the fused protein LPD-MAGE3-His is immunogenicin mice, and that this immunogenicity (the proliferative response andantibody response) can be further increased by the use of the adjuvantdescribed above. Purification can be enhanced by derivatising the thiolsthat form disulphide bonds.

We have also demonstrated that a better antibody response was triggeredby the vaccination with the LPD-MAGE-3-His in the presence of theadjuvant. The predominant isotype found in the serum of C57BL/6 beingIgG2b suggesting that a TH1 type immune response was raised.

In the human, clinical setting a patient treated with LPD-MAGE3-His inan unadjuvanted formulation was cleared of melanoma.

REFERENCES

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1. A process for the production of an immunogenic polypeptide comprisingthe steps of: (i) treating a polypeptide consisting of SEQ ID NO:2 toreduce a disulfide bond and produce a free thiol group; (ii) blockingsaid free thiol group with a blocking agent; (iii) purifying apolypeptide resulting from step (ii) using one or more chromatographicpurification steps, where said blocking agent is not removed from saidthiol group.
 2. The process of claim 1, wherein said blocking agent isiodoacetamide.
 3. The process of claim 1, wherein the resultingimmunogenic polypeptide is carboxyamidated.
 4. The process of claim 1,wherein the resulting immunogenic polypeptide is carboxymethylated. 5.The process of claim 1, wherein the resulting immunogenic polypeptide islipidated.
 6. The process of claim 1, further comprising formulating theresulting immunogenic polypeptide as a pharmaceutical composition,wherein said blocking agent is not removed from said thiol group.
 7. Aprocess for the production of an immunogenic composition comprising thesteps of: (i) treating a polypeptide consisting of SEQ ID NO:2 to reducea disulfide bond and produce a free thiol group; (ii) blocking said freethiol group with a blocking agent; (iii) purifying a polypeptideresulting from step (ii) using one or more chromatographic purificationsteps, where said blocking agent is not removed from said thiol group;and (iv) formulating a polypeptide resulting from step (iii) as apharmaceutical composition, wherein said blocking agent is not removedfrom said thiol group.
 8. The process of claim 7, wherein said blockingagent is iodoacetamide.
 9. The process of claim 7, wherein the resultingpolypeptide is carboxyamidated.
 10. The process of claim 7, wherein theresulting polypeptide is carboxymethylated.
 11. The process of claim 7,wherein the resulting polypeptide is lipidated.